EP0290630A1 - 9-substituted guanines - Google Patents

Abstract

New guanine compounds substituted in position 9 have the formula (I) where R 'is acetyl, hydrogen; R '' is 2-tetrahydrofuryl, 2-tetrahydropyranyl. These compounds have anti-viral properties.

Description

Technical field

The present invention relates to organic chemistry and relates in particular to new compounds, namely 9-substituted guanines. The compounds given have an antiviral activity.

Current state of the art

welches als Wirkstoff des Arzneimittels Zovirax (Azyklovir), hergestellt von der Firma "The Wellcome Foundation Ltd.", London (DE, A, Nr. 2539963) dient.Various compounds are known which are active against viruses, for example 9- (2-hydroxyethoxy methyl) guanine of the following formula

which serves as an active ingredient of the drug Zovirax (azyklovir), manufactured by the company "The Wellcome Foundation Ltd.", London (DE, A, No. 2539963).

Zovirax is an effective anti-virus preparation of the second generation that inactivates herpes simplex viruses HSV-1 and HSV-2 as well as zoster viruses and other viruses in humans and animals.

The preparation is sparingly water-soluble (2 mg / ml), and thus the nephrotoxicity should be noted when administered orally.

The preparation is administered intravenously as a longer (two-hour) infusion. Due to its low solubility, the preparation cannot be administered intramuscularly, and in the event of accidental intravenous administration, the preparation that has entered the surrounding tissue causes necrosis due to the high alkaline reaction of the solution for injection (pH = 11.0).

Disclosure of the invention

The claimed compounds are new and are not described in the specialist literature.

The invention is based, new task To produce compounds which have an antiviral activity and have an increased solubility in water and lipophilic substances of the organism.

haben, worin

• R' für Azetyl, Wasserstoff,
• R" für 2-Tetrahydrofuryl oder 2-Tetrahydropyranyl stehen.

The object is achieved in that the claimed new compounds are 9-substituted. Guanine according to the invention the general formula

have what

• R 'for acetyl, hydrogen,
• R "stand for 2-tetrahydrofuryl or 2-tetrahydropyranyl.

The compounds according to the invention are crystalline substances, the structure of which is demonstrated by the entirety of the physicochemical properties (Tables 1 and 2).

The best embodiment of the invention

The antiviral activity of the compounds according to the invention in animals was compared to zovirax (azyklovir). The tests were carried out on the model of experimental herpetic encephalitis in non-purebred white mice. Mice weighing 8 to 10 g were infected with the herpes simplex yirus of the first type. The mice were infected in two ways, intraperitoneally at a dose exceeding ₅₀ times LD ₅₀ and intracerebrally at a dose of 10 to 30 LD ₅₀ . The substances were administered as aqueous solutions or suspensions with Tween 80 intravenously and orally. The following methodology was used: the first injection was carried out 3 hours after infection by the virus and then from the 2nd to the 7th day, with intravenous administration, the compounds were introduced once a day, and with oral: application, the daily dose was halved and injected twice a day . The daily doses were between 0.01 and 200 mg / kg. The unit dose was dissolved in 0.1 ml of distilled water for oral administration and in 0.2 ml for intravenous administration.

The corresponding volume of the isotonic saline solution was applied to the mice from the control group.

The effectiveness of the compounds was assessed after the reduction in lethality in animals to which the compounds according to the invention and Zovirax (comparative preparation) were administered, in comparison with the control group. The test results are shown in Tables 3 and 4.

As can be seen from Table 3, 9- / 2- (2-tetrahydrofuryloxy) ethoxymethyl / -N ^2- e.zetylgua- nin reduces mortality in mice both in the case of oral administration and intravenous injection. The effectiveness is the same in both cases. Perhaps this is due to the fact that the compound was administered orally twice a day and intravenously once a day in the same daily dose. When the compound was applied twice within 24 hours, the effectiveness of the oral therapy was increased and, in doing so, that of the intravenous therapy was brought up. As the table shows, the activity of the compound according to the invention (the maximum reduction in mortality by 75%) is higher than that of Zovirax (the reduction in mortality by 65.9%). In the course of the similar experiments, the effectiveness of 9- / 2- (2-tetrahydrofuryloxy) ethoxymethyl / guanine was approximately 63%, that of 9- / 2- (2-tetrahydrofyranyloxy) ethoxymethyl / guanine was approximately 58% and that of 9- /2-(2-etrahydropyranyloxy)äthoxymethyl/_.N2.:acetylguanine about 15%.

The effectiveness of 9- / 2- (2-tetrahydrofuryloxy) athoxymethyl / -N ² -acetylguanine has also been tested in mice infected by herpes simplex virus intracerebrally, ie by direct administration of virus into the brain. The virus dose was 10 to 30 times as large as LD ₅₀ (the test results are shown in Table 4). The compounds were administered orally and intravenously according to the methodology given in the previous experiment.

As can be seen from Table 4, the compound according to the invention is effective in a wider dose range.

When the compound according to the invention was administered orally, the maximum effect (the reduction in the lethality by 56.3% compared to the control) was noted when the daily dose of 100 or 200 mg / kg was introduced. Zovirax, administered in a daily dose of 100 mg / kg and 200 mg / kg, reduced the lethality by 33.3 and only by 26.2%. The efficacy of the two compounds was similar at lower doses (from 0.1 to 10 mg / kg).

In the intravenous injection of the compound of the invention in a dose of 10 mg / kg mortality reduced by 55.8%, which corresponds to the effect of Zovirax when applied in the same dose. However, with Zovirax you cannot increase your dose without changing the pH of the solution because the solubility of Zovirax is limited. If 9- / 2- (2-tetrahydrofuryloxy) ethymethyl / -N2_acetylguanine is used, its dose can increase to 100 mg / kg. This reduces the lethality by 66.5%. The breadth of the pharmacological effect in the compound according to the invention is 10 times higher than in the Zovirax.

The number of plates was counted in the course of in vitro experiments using the single-layer culture of the primary fibroblasts of chicken embryos infected by type I herpes simplex virus. The virus titer was reduced by more than 1.5 IgBFU / ml when used. 9- / 2- (2-tetrahydrofuryloxy) ethoxymethyl / -N2-acetylguanine, 9- / 2_ (2-tetrahydrofuryloxy) -athoxymethyl / -guanine and 9- / 2- (2-tetrahydropyranyloxy) ethoxymethyl / guanine were found at a concentration of 100 µg / ml, while in the case of the application of 9- / 2- (2-tetrahydropyranyloxy) ethoxymethyl / _N ^2- acetylguanine at a concentration of 400 µg / ml. These concentrations are significantly higher than the minimum effective concentration of Zovirax. Since in vivo experiments show a high antiviral activity of the compounds according to the invention, it can be assumed that they are activated in the organism, ie they act as transport forms in that they are converted into the active compound in the organism. This can probably be explained by the fact that the overall effect of the compounds according to the invention on the organism of animals is less than that of Zovirax:

-Tetrshydrofuryloxy)äthoxyme thyl /-N²-azaetylgtzanin 3300 mg/kg, bei 9-/2-(2-Tetrahydropyranyloxy)ätho- xymethyl/guinin übersteigt LD50 2000 mg/kg und ist 1800 mg/kg bei Zovirax gleich.For example, LD ₅₀ for mice with a single intraperitoneal injection of 9- / 2- (2-

-Tetrshydrofuryloxy) äthoxyme thyl / -N ² -azaetylgtzanin 3300 mg / kg, with 9- / 2- (2-tetrahydropyranyloxy) äthoxymethyl / guinine exceeds LD50 2000 mg / kg and is equal to 1800 mg / kg with Zovirax.

The compounds according to the invention have better solubility in water than Zovirax. 9- / 2- (2-tetrahydrofuryloxy) ethoxymethyl / -N -acetylguanine is particularly soluble in water, its solubility is 120 mg / ml and exceeds the solubility of Zovirax (2 mg / ml) by 60 times.

The solubility of 9- / 2- (2-tetrahydropyranyloxy) ethoxymethyl / -N ^2_ acetylguanine is also approximately 100 mg / ml. The solubility of 9-12- (2-tetrahydrofuryloxy) -ethoxymethyl / guanine and 9- / 2- (2-tetrahydropyranyloxy) -ethoxymethyl / guanine is 2.5 to 3 mg / ml. The compounds according to the invention are also better soluble than zovirax in weakly polar organic solvents. For example, the solubility of 9- / 2- (2-tetrahydrofuryloxy) a, thoxymethyl / -N ² -acetylguanine in chloroform and that of Zovirax is 10 mg / ml or below 1 mg / ml. Daraux can be assumed that 9- / 2- (2-tetrahydrofuryloxy) -ethoxymethyl / -N ^2- acetylguanine in lipophilic substances of the organism, including in ganglia of sensitive nerves, where the accumulation of virus particles takes place in the latency period of the disease , can penetrate deeper.

The compounds of the invention are stable in neutral aqueous solutions (pH of about 7.4), but are hydrolyzed in an acidic medium at a pH of less than.

• a) eine bedeutend höhere (60fache) Wasserlöslichkeit, welche die Lösungen von 9-/2-(2-Tetrahydrofuryloxy)-athoxymethyl/-N -azetylguanin für intravenöse und intraperitoneale Injektionen zu verwenden ermöglicht;
• b) eine geringere Nephrotoxizität und als Folge daraus eine Möglichkeit, in den Organismus höhere (19fache) Dosen einzuführen, was eine größere Herabsetzung der Letalität, bedingt durch herpetische Enzephalitis von Mäusen (um 66,5 % bei intrazerebraler Einführung von Virus und intravenöser Injektion der Verbindung und um 55 % bei Azyklovir) ergibt.
• c) eine vollständigere Löslichkeit in schwach polaren organischen Lösungsmitteln (Azyklovir ist darin unlöslich), was das Eindringen in Ganglien sensibler Nerven ermöglicht, wo die Ansammlung von Virusteilchen in der Latenzzeit der Krankheit erfolgt.

The results obtained show the advantages of the compounds according to the invention over zovirax (azyklovir):

• a) a significantly higher (60 times) water solubility, which the solutions of 9- / 2- (2-tetrahydrofuryloxy) - allows to use athoxymethyl / -N -acetylguanine for intravenous and intraperitoneal injections;
• b) lower nephrotoxicity and, as a result, the possibility of introducing higher (19-fold) doses into the organism, which results in a greater reduction in lethality due to herpetic encephalitis in mice (by 66.5% with intracerebral introduction of the virus and intravenous injection of the Compound and by 55% for azyklovir).
• c) more complete solubility in weakly polar organic solvents (acyclovir is insoluble in it), which allows penetration into sensitive nerve ganglia, where virus particles accumulate during the latency period of the disease.

The compounds of the invention are prepared as follows.

9- (2-Acetoxyathoxymethyl) -N -acetylguanine is introduced into a flask equipped with a stirrer and the water is added. The suspension obtained is stirred and cooled to a temperature between 0 and minus 5 ° C., then the cooled 2 n-NaOH solution is added. The contents of the flask are stirred at the same temperature for a further 1 hour and neutralized with acetic acid until a pH of 6 to 7 is achieved. As a result, a solution is obtained which is kept within 24 hours at a temperature between 0 and minus 5 ° C. The precipitate of 9- (2-hydroxyethyloxymethyl) -N ² -acetylguanine is filtered off, washed and dried.

The 9- (2-hydroxyethoxymethyl) -N ² _ acetylguanine obtained is dissolved in dimethylformamide at a temperature of 50 to 60 ° C. The solution is cooled to a temperature between 0 and minus 5 ° and saturated with hydrogen chloride at this temperature. After this, stir with stirring 2,3- -Dihydrofuran or 2,3-dihydropyran too. The reaction mixture is blurred within 3 hours at a temperature between 0 and minus 5 ° C, after which it is neutralized with triethylamine. The precipitate of triethylamine hydrochloride is filtered off. The filtrate is evaporated in vacuo and the backbone crystallizes from ethyl acetate and then from ethanol. 9- / 2- (2-tetrahydrofuryloxy) ethoxymethyl / -N ² , zetylguanine or, if 2,3-dihydropyran instead of 2,3-dihydrofuran is used, 9- / 2- (2-tetrahydropyranyloxy) ethaxymethyl / -N ² -acetylguanine.

An aqueous solution of methylamine is added to the 9- / 2- (2-tetrahydrofuryloxy) - ethoxymeth.yl / -N ² -acetylguanine prepared, mixed within 2 hours at room temperature and then evaporated in vacuo. The residue is recrystallized from aqueous ethanol.

9- / 2-tetrahydrofuryloxy) athoxymethyl / guanine is obtained.

Treating 9- / 2- (2-tetrahydropyrsnyloxy) ethoxy-methyl / -N ² -acetylguanine with aqueous methylamine solution gives 9- / 2- (2-tetrahydropyranyloxy) ethoxymethyl / guanine in a similar manner.

To better understand the present invention, the following specific examples are given.

example 1

3.0 g (96 mmol) of 9- (2-acetoxymethyl) Iff2-acetyl guanine and 60 ml of water are introduced into a flask equipped with a stirrer. The resulting suspension is cooled to 0 ° C. and 60 ml of 2 n-NaOH solution, which has been cooled to 0 ° C. in advance, are added with stirring. The reaction mixture is stirred at a temperature between 0 and minus 5 ° C within 1 hour and then neutralized with acetic acid until a pH of 6 to 7 is achieved. After cooling within 24 hours, the precipitate is precipitated filtered off, washed with a small amount of cold water and dried. 2.18 g (84%) of 9- (2-hydroxyathoxymethyl) -N -acetylguanine are obtained with a melting point of 212 ° to 214 ° C., R _f = 0.21 (in a chloroform-methanol system at a ratio of 10: 2) .

1.20 g (4.5 mmol) of 9- (2-hydroxyethoxymethyl) -N ² -acetylguanine, prepared as described above, is introduced into a flask equipped with a stirrer and at a temperature between 50 and 60 ° C. in 40 ml Dissolved dimethylformamide. The solution is cooled to a temperature between 0 and 5 ° C and saturated at this temperature with dry hydrogen chloride. Then 4 ml of 2,3-dihydrofuran are gradually added to the reaction mixture with stirring and cooling to 0.degree. The solution is mixed within 3 hours at a temperature between 0 and minus 5 ° C and then the excess hydrogen chloride is neutralized with triethylamine.

The precipitated precipitate of triethylamine hydrochloride is filtered off and washed with a small amount of dimethylformamide. The filtrate is evaporated in vacuo. The residue is recrystallized from dry ethyl acetate and then from ethanol. 1.26 g (82.9%) of 9- / 2- (2-tetrahydrofuryloxy) athoxymethyl / -N ² -acetylguanine are prepared.

The physico-chemical characteristics that demonstrate the structure of the compound are summarized in Tables 1 and 2 above.

Example 2

Into a flask equipped with a stirrer flask is placed _{1, 1} 8 g (3.5 mmol) 9- / 2- (2-tetrahydrofuryloxy) ethoxy methyl / -N2-azetylguanin, hergestelslt as described in Example 1 a. 20 ml of a 25% aqueous solution of methylamine are added. The reaction mixture is stirred at room temperature within 2 hours and then evaporated in vacuo at a temperature not exceeding 30 ° C. The dry residue is recrystallized twice from aqueous ethanol. 0.87 g (84%) of 9- / 2- (2-tetrahydrofuryloxy) ethoxymethyl / guanine are obtained.

Example 3

1.20 g (4.5 mmol) of 9- (2-hydroxyathoxymethyl) -N ² -acetylguanine, prepared as described in Example 1, are introduced into a flask provided with a stirrer. 40 ml of dimethylformamide are added and the mixture is heated at a temperature of 50 to 60 ° C. until the precipitate is completely dissolved. The solution obtained is cooled to a temperature between 0 and minus 5 ° C and saturated with dry hydrogen chloride at this temperature. Then 4 ml of 2,3-dihydropyran are gradually added to the reaction mixture while stirring and cooling to 0 ° C., after which the excess hydrogen chloride is neutralized by adding triethylamine. The precipitate of triethylamine hydrochloride is filtered off and the filtrate is evaporated in vacuo. The residue is recrystallized from dry ethyl acetate and then from ethanol. 0.89 g (56.5%) of 9- / 2- (2-tetrahydropyranyl oxy) ethoxymethyl / -N ² -acetylguanine are obtained.

Example 4

1.23 g (3.5 mmol) of 9- / 2- (2-tetrahydropyranyloxy) ethoxymethyl / -N ² -acetylguanine, prepared as described in Example 3, are introduced into a flask provided with a stirrer. 20 ml of 25% aqueous solution of methylamine are added. The reaction mixture is stirred at room temperature within 2 hours and then evaporated in vacuo at a temperature not exceeding 30 ° C. The dry residue is recrystallized twice from aqueous ethanol. 0.86 g (80%) of 9- / 2- (2-tetra- hydropyranyloxy) ethoxymethyl / guanine.

The physicochemical properties of the compounds prepared in Examples 1 to 4, which demonstrate their structure, are summarized in Tables 1 and 2.

Industrial applicability

The compounds according to the invention can be used in medicine.

Claims (1)

9-substituted guanines of the general formula

where R 'is acetyl, hydrogen and R "is 2-tetrahydrofuryl, 2-tetrahydropylanyl.